Reinforced placental tissue grafts and methods of making and using the same

ABSTRACT

Described herein are tissue grafts derived from the placental tissue that are reinforced with at least one biocompatible mesh. The tissue grafts possess good adhesion to biological tissues and are useful in wound healing applications. Also described herein are methods for making and using the tissue grafts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/683,699, filed on Aug. 15, 2012 and U.S. Provisional ApplicationSer. No. 61/808,171 filed on Apr. 3, 2013, the entirety of both whichare incorporated herein by reference.

BACKGROUND

Human placental membrane (e.g. amniotic membrane) has been used forvarious types of reconstructive surgical procedures since the early1900s. However, the physical attributes of placental allografts do limittheir use. For example, placental allografts cannot be sutured, limitingtheir utility with clinicians who feel suturing prevents micro movementwhich can disrupt the clot and subsequent blood supply to the graftedarea, or prefer to first tack the barrier membrane in place and then addthe bone graft. Placental allografts, traditional cadaveric allograft,and xenograft collagen barrier membranes are adaptable and conformable;however, they possess inadequate tensile strength and stiffness tostabilize grafted bone in alveolar horizontal and/or vertical boneaugmentations.

SUMMARY

Described herein are tissue grafts derived from the placental tissuethat are reinforced with at least one biocompatible mesh. The tissuegrafts possess good adhesion to biological tissues and are useful inwould healing applications. Also described herein are methods for makingand using the tissue grafts.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an overview flow chart of the process for making the tissuegrafts described herein.

FIG. 2 is a perspective view of an exemplary drying fixture for makingthe tissue grafts described herein.

FIG. 3 depicts several embodiments of the reinforced tissue graftsdescribed herein.

FIG. 4 shows an exemplary drying fixture and drying rack useful inpreparing tissues grafts described herein.

FIG. 5 shows the application of a reinforced tissue graft over atrephine defect (9), orbital defect (10), repair of a sinus (11),maxillary vertical and horizontal bone augmentation (12), and mandibularvertical and horizontal bone augmentation (13).

FIG. 6 shows the application of a reinforced tissue graft in a segmentallong bone defect (14).

FIG. 7 shows the application of a reinforced tissue graft as arterialstent (15).

FIG. 8 shows a forward perspective view of a dehydration device asdescribed herein.

FIG. 9 shows an overhead perspective view of a dehydration device asdescribed herein.

FIG. 10 shows a side perspective view of a dehydration device asdescribed herein.

FIG. 11 shows a back perspective view of a dehydration device asdescribed herein. FIGS. 8-11 are sometimes referred to as FIGS. 90-93,respectively.

DETAILED DESCRIPTION

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, synthetic methods, or uses as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a cross-linking agent” includes mixtures of two or moresuch agents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” as used herein is any vertebrate organism includingmammals such as domesticated animals and primates such humans.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

The term “placental tissue” refers to any and all of the well-knowncomponents of the placenta including but not limited to amnion, chorion,Wharton's Jelly, and the like. In one preferred embodiment, theplacental tissue does not include any of the umbilical cord components(e.g., Wharton's jelly, umbilical cord vein and artery, and surroundingmembrane).

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

I. Reinforced Tissue Grafts and Methods for Making Thereof

Described herein are reinforced tissue grafts derived from the placentathat possess good adhesion to biological tissues and are useful in wouldhealing applications. FIG. 1 depicts an exemplary overview (100) andcertain aspects of the steps to harvest, process, and prepare placentalmaterial for later use as a tissue graft. More detailed descriptions anddiscussion regarding each individual step will follow. Initially, theplacenta tissue is collected from a consenting patient following anelective Cesarean surgery (step 110). The material is preserved andtransported in conventional tissue preservation manner to a suitableprocessing location or facility for check-in and evaluation (step 120).Gross processing, handling, and separation of the amnion and chorionthen takes place (step 130). Acceptable tissue is then decontaminated(step 140), followed by the optional steps of substantially removing theepithelium layer from the placental tissue (e.g., amnion or Wharton'sjelly) to expose the basement membrane (step 145) and cross-linking ofthe placental tissue(s) (step 147) used to prepare the reinforced tissuegrafts. The reinforced tissue graft is then prepared from the placentaltissue and the graft is subsequently dehydrated (step 150), cut andpackaged (step 160), sterilized using gamma radiation or electron beamradiation (step 165), and released (step 170) to the market for use bysurgeons and other medical professionals in appropriate surgicalprocedures and for wound care. Each step is described in detail below.

Initial Tissue Collection (Step 110)

The components used to produce the tissue grafts are derived from theplacenta. The source of the placenta can vary. In one aspect, theplacenta is derived from a mammal such as human and other animalsincluding, but not limited to, cows, pigs, and the like can be usedherein. In the case of humans, the recovery of the placenta originatesin a hospital, where it is collected during a Cesarean section birth.The donor, referring to the mother who is about to give birth,voluntarily submits to a comprehensive screening process designed toprovide the safest tissue possible for transplantation. The screeningprocess preferably tests for antibodies to the human immunodeficiencyvirus type 1 and type 2 (anti-HIV-1 and anti-HIV-2), antibodies to thehepatitis B virus (anti-HBV) hepatitis B surface antigens (HBsAg),antibodies to the hepatitis C virus (anti-HCV), antibodies to the humanT-lymphotropic virus type I and type II (anti-HTLV-I, anti-HTLV-II),CMV, and syphilis, and nucleic acid testing for human immune-deficiencyvirus type 1 (HIV-1) and for the hepatitis C virus (HCV), usingconventional serological tests. The above list of tests is exemplaryonly, as more, fewer, or different tests may be desired or necessaryover time or based upon the intended use of the grafts, as will beappreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta is collected prior to the completion of obtaining theresults from the screening tests and delivery cultures, such tissue islabeled and kept in quarantine. The placenta is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied and obtains final approval from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the tissue. The sterile shipmentbag containing the tissue is then stored in a refrigerator until readyfor further processing.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placental tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a controlled environment. In one aspect, the placentais processed at room temperature. If the controlled environment is amanufacturing hood, the sterile supplies are opened and placed into thehood using conventional sterilization techniques. If the controlledenvironment is a clean room, the sterile supplies are opened and placedon a cart covered by a sterile drape. All the work surfaces are coveredby a piece of sterile drape using conventional sterilization techniques,and the sterile supplies and the processing equipment are placed ontothe sterile drape, again using conventional sterilization techniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hyperisotonic salinesolution (e.g., 18% NaCl) that is at room or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placental tissue to reach room temperature, which facilitates theseparation of the placental components from each other (e.g., amnionmembrane and chorion). After having warmed up to ambient temperature(e.g., after about 10-30 minutes), the placenta is then removed from thesterile processing basin and laid flat on a processing tray with theamnion membrane layer facing down for inspection.

The placenta is examined for discoloration, debris or othercontamination, odor, and signs of damage. The size of the tissue is alsonoted. A determination is made, at this point, as to whether the tissueis acceptable for further processing.

The amnion and chorion are next carefully separated. In one aspect, thematerials and equipment used in this procedure include a processingtray, 18% saline solution, sterile 4×4 sponges, and two sterile Nalgenejars. The placenta tissue is then closely examined to find an area(typically a corner) in which the amnion can be separated from thechorion. The amnion appears as a thin, opaque layer on the chorion.

The fibroblast layer is identified by gently contacting each side of theamnion with a piece of sterile gauze or a cotton tipped applicator. Thefibroblast layer will stick to the test material. The amnion is placedinto processing tray basement membrane layer down. Using a bluntinstrument, a cell scraper, or sterile gauze, any residual blood is alsoremoved. This step must be done with adequate care, again, so as not totear the amnion. The cleaning of the amnion is complete once the amnionis smooth and opaque-white in appearance.

The methods described herein do not remove all cellular components inthe amnion. This technique is referred to in the art as“decellularization.” Decellularization generally involves the physicaland/or chemical removal of all cells present in the amnion, whichincludes epithelial cells and fibroblast cells. For example, althoughthe removal of epithelial cells is optional, the fibroblast layerpresent in the amnion stromal layer is intact, even if the intermediatetissue layer is removed. Here, fibroblast cells are present in thefibroblast layer.

In certain aspects, the intermediate tissue layer, also referred to asthe spongy layer, is substantially removed from the amnion in order toexpose the fibroblast layer. The term “substantially removed” withrespect to the amount of intermediate tissue layer removed is definedherein as removing greater than 90%, greater than 95%, or greater than99% of the intermediate tissue layer from the amnion. This can beperformed by peeling the intermediate tissue layer from the amnion.Alternatively, the intermediate tissue layer can be removed from theamnion by wiping the intermediate tissue layer with gauze or othersuitable wipe. The resulting amnion can be subsequently decontaminatedusing the process described below. Not wishing to be bound by theory,the removal of the intermediate layer can accelerate the drying of thetissue graft, particularly if multiple amnion membranes are used toproduce the graft. The intermediate layer can be removed from the amnionprior contacting the amnion with the cross-linking agent or, in thealternative, can be removed after the amnion has been contacted with thecross-linking agent.

When the placental tissue is Wharton's jelly, the following exemplaryprocedure can be used. Using a scalpel or scissors, the umbilical cordis dissected away from the chorionic disk. Once the veins and the arteryhave been identified, the cord is dissected lengthwise down one of theveins or the artery. Once the umbilical cord has been dissected,surgical scissors and forceps can be used to dissect the vein and arterywalls from the Wharton's jelly. Next, the outer layer of amnion isremoved from the Wharton's jelly by cutting the amnion. Here, the outermembrane of the umbilical cord is removed such that Wharton's jelly isthe only remaining component. Thus, the Wharton's jelly as used hereindoes not include the outer umbilical cord membrane and umbilical cordvessels. The Wharton's jelly can be cut into strips. In one aspect, thestrips are approximately 1-4 cm by 10-30 cm with an approximatethickness of 1.25 cm; however, other thicknesses are possible dependingon the application.

Chemical Decontamination (Step 140)

The amnion and chorion isolated above can be chemically decontaminatedusing the techniques described below. In one aspect, the amnion andchorion is decontaminated at room temperature. In one aspect, the amnionproduced in step 130 can be placed into a sterile Nalgene jar for thenext step. In one aspect, the following procedure can be used to cleanthe amnion. A Nalgene jar is aseptically filled with 18% salinehypertonic solution and sealed (or sealed with a top). The jar is thenplaced on a rocker platform and agitated for between 30 and 90 minutes,which further cleans the amnion of contaminants. If the rocker platformwas not in the critical environment (e.g., the manufacturing hood), theNalgene jar is returned to the controlled/sterile environment andopened. Using sterile forceps or by aseptically decanting the contents,the amnion is gently removed from the Nalgene jar containing the 18%hyperisotonic saline solution and placed into an empty Nalgene jar. Thisempty Nalgene jar with the amnion is then aseptically filled with apre-mixed antibiotic solution. In one aspect, the premixed antibioticsolution is composed of a cocktail of antibiotics, such as StreptomycinSulfate and Gentamicin Sulfate. Other antibiotics, such as Polymixin BSulfate and Bacitracin, or similar antibiotics now available oravailable in the future, are also suitable. Additionally, it ispreferred that the antibiotic solution be at room temperature when addedso that it does not change the temperature of or otherwise damage theamnion. This jar or container containing the amnion and antibiotics isthen sealed or closed and placed on a rocker platform and agitated for,preferably, between 60 and 90 minutes. Such rocking or agitation of theamnion within the antibiotic solution further cleans the tissue ofcontaminants and bacteria. Optionally, the amnion can be washed with adetergent. In one aspect, the amnion can be washed with 0.1 to 10%, 0.1to 5%, 0.1 to 1%, or 0.5% Triton-X wash solution.

If the rocker platform was not in the critical environment (e.g., themanufacturing hood), the jar or container containing the amnion andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the amnion is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The amnion is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The amnion may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the amnion is ready to be dehydratedand processed further.

In the case of chorion, the following exemplary procedure can be used.After separation of the chorion from the amnion and removal of clottedblood from the fibrous layer, the chorion is rinsed in 18% salinesolution for 15 minutes to 60 minutes. During the first rinse cycle, 18%saline is heated in a sterile container using a laboratory heating platesuch that the solution temperature is approximately 48° C. The solutionis decanted, the chorion tissue is placed into the sterile container,and decanted saline solution is poured into the container. The containeris sealed and placed on a rocker plate and agitated for 15 minutes to 60minutes. After 1 hour agitation bath, the chorion tissue was removed andplaced into second heated agitation bath for an additional 15 minutes to60 minutes rinse cycle. Optionally, the chorion tissue can be washedwith a detergent (e.g., Triton-X wash solution) as discussed above forthe decontamination of amnion. The container is sealed and agitatedwithout heat for 15 minutes to 120 minutes. The chorion tissue is nextwashed with deionized water (250 ml of DI water×4) with vigorous motionfor each rinse. The tissue is removed and placed into a container of1×PBS w/EDTA solution. The container is sealed and agitated for 1 hourat controlled temperature for 8 hours. The chorion tissue is removed andrinsed using sterile water. A visual inspection was performed to removeany remaining discolored fibrous blood material from the chorion tissue.The chorion tissue should have a cream white visual appearance with noevidence of brownish discoloration.

The following exemplary procedure can be used when the placental tissueis Wharton's jelly. The Wharton's jelly is transferred to a sterileNalgene jar. Next, room temperature 18% hypertonic saline solution isadded to rinse the tissue and the jar is sealed. The jar is agitated for30 to 60 minutes. After incubation, the jar is decontaminated andreturned to the sterile field. The tissue is transferred to a cleansterile Nalgene jar and prewarmed (about 48° C.) with 18% NaCl. Thecontainer is sealed and placed on rocker plate and agitated for 60 to 90minutes.

After the rinse, the jar is decontaminated and returned to the sterilefield. The tissue is removed and placed into an antibiotic solution. Thecontainer is sealed and agitated for 60 to 90 minutes on a rockerplatform. Following incubation, the jar may be refrigerated at 1 to 10°C. for up to 24 hours.

The Wharton's jelly is next transferred to a sterile basin containingapproximately 200 mL of sterile water. The tissue is rinsed for 1-2minutes and transferred to a sterile Nalgene jar containingapproximately 300 ml of sterile water. The jar is sealed and placed onthe rocker for 30 to 60 minutes. After incubation, the jar is returnedto the sterile field. The Wharton's jelly should have a cream whitevisual appearance with no evidence of brownish discoloration.

Removal of Epithelium Layer from Placental Tissue (Step 145)

In certain aspects, it is desirable, although optional, to remove theepithelium layer present on the placental tissue. In one aspect, theepithelium layer present on the amnion is substantially removed in orderto expose the basement layer of the amnion. In another aspect, theepithelium layer present on the Wharton's jelly is substantiallyremoved. The term “substantially removed” with respect to the amount ofepithelium removed is defined herein as removing greater than 90%,greater than 95%, or greater than 99% of the epithelial cells from theamnion. The presence or absence of epithelial cells remaining on theamnion layer can be evaluated using techniques known in the art. Forexample, after removal of the epithelial cell layer, a representativetissue sample from the processing lot is placed onto a standardmicroscope examination slide. The tissue sample is then stained usingEosin Y Stain and evaluated as described below. The sample is thencovered and allowed to stand. Once an adequate amount of time has passedto allow for staining, visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step and thebefore the tissue has been dehydrated as described in the next section.For example, a representative sample graft is removed for microscopicanalysis. The tissue sample is place onto a standard slide, stained withEosin Y and viewed under the microscope. If epithelium is present, itwill appear as cobblestone-shaped cells.

The methods described herein, particularly steps 130 and 145, do notremove all cellular components in the amnion. This technique is referredto in the art as “decellularization.” Decellularization generallyinvolves the physical and/or chemical removal of all cells present inthe amnion, which includes epithelial cells and fibroblast cells.Although step 145 does remove epithelial cells, the fibroblast layerpresent in the amnion stromal layer is intact (i.e., includes fibroblastcells), even after removal of the intermediate layer discussed in step130.

Cross-Linking Step (step 147)

Depending upon the application of the tissue graft, one or moreplacental tissues use to produce the reinforced tissue graft can beoptionally cross-linked. Not wishing to be bound by theory, thecross-linking of the placental tissue can modify the resorptionproperties of the placental tissue. For example, the placental tissuecan be cross-linked in order to regulate the rate of release of growthfactors present in the placental tissue. In other aspects, thecross-linked placental tissue can be sufficiently cross-linked in orderto prevent bioactive agents (e.g., INFUSE®) from leaching out of thereinforced tissue graft. Here, the cross-linked placental tissue acts asa barrier.

The placental tissue grafts can be cross-linked using a number oftechniques. In one aspect, cross-linking may be achieved by chemical,thermal, radiation, fibronectin, fibrinogen and/or hydrogelcross-linking methods. In other aspects, the placental tissue can beindividually treated with a cross-linking agent prior to lamination andformation of the reinforced tissue graft. In general, the cross-linkingagent is nontoxic and non-immunogenic. When two or more placentaltissues are treated with the cross-linking agent, the cross-linkingagent can be the same or different. In one aspect, the chorion andamnion can be treated separately with a cross-linking agent or, in thealternative, the chorion and amnion can be treated together with thesame cross-linking agent. In certain aspects, the amnion or chorion canbe treated with two or more different cross-linking agents.

The conditions for treating the placental tissue can vary. In oneaspect, the amnion or chorion can be placed in a container holding anaqueous solution of the cross-linking agent. In one aspect, theconcentration of the cross-linking agent is from 0.1 M to 5 M, 0.1 M to4 M, 0.1 M to 3 M, 0.1 M to 2 M, or 0.1 M to 1 M. In another aspect, theplacental tissue is treated with the cross-linking agent for 1 to 2seconds up to 60 minutes. In a further aspect, the amnion or chorion aretreated with the cross-linking agent at room temperature up to 50° C.

The cross-linking agent generally possesses two or more functionalgroups capable of reacting with proteins to produce covalent bonds. Inone aspect, the cross-linking agent possesses groups that can react withamino groups present on the protein. Examples of such functional groupsinclude, but are not limited to, hydroxyl groups, substituted orunsubstituted amino groups, carboxyl groups, and aldehyde groups. In oneaspect, the cross-linker can be a dialdehydes such as, for example,glutaraldehyde. In another aspect, the cross-linker can be acarbodiimide such as, for example,(N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC). In otheraspects, the cross-linker can be an oxidized dextran, p-azidobenzoylhydrazide, N-[alpha-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[beta-(4-azidosalicylamido)ethyl]disulfide,bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate,disuccinimidyl suberate, and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, abifunctional oxirane (OXR), or ethylene glycol diglycidyl ether (EGDE).

In one aspect, sugar is the cross-linking agent, where the sugar canreact with proteins present in the placental tissue to form a covalentbond. For example, the sugar can react with proteins by the Maillardreaction, which is initiated by the nonenzymatic glycosylation of aminogroups on proteins by reducing sugars and leads to the subsequentformation of covalent bonds. Examples of sugars useful as across-linking agent include, but are not limited to, D-ribose,glycerose, altrose, talose, ertheose, glucose, lyxose, mannose, xylose,gulose, arabinose, idose, allose, galactose, maltose, lactose, sucrose,cellibiose, gentibiose, melibiose, turanose, trehalose, isomaltose, orany combination thereof. Thus, in one aspect, the amnion or chorioninclude at least one cross-linker covalently attached to the membrane.In another aspect, a tissue graft includes an amnion and a chorionlaminate, wherein the amnion and chorion are covalently attached to oneanother via a cross-linker.

The following procedure provides an exemplary method for treating theamnion and chorion with a cross-linking agent. The cleaned anddecontaminated chorion and amnion are placed on the sterile field in themanufacturing hood. The tissue is transferred to a Nalgene jarcontaining a cross-linking agent, preferably 0.05 to 1 M D-ribose,preferably 0.2 M (3.01%) D-ribose, for 1 to 60 minutes, preferably 5minutes. The tissues may be treated with the cross-linking agent eitherin separate containers or together in the same container. After theincubation, the tissue is removed from the solution and, optionally,allowed to dry.

Preparation of Micronized Compositions and Pharmaceutical CompositionsThereof

Once the placental tissue or components thereof as described above havebeen dehydrated individually or in the form of tissue graft, thedehydrated tissue(s) is micronized. The micronized compositions can beproduced using instruments known in the art. For example, the RetschOscillating Mill MM400 can be used to produce the micronizedcompositions described herein. The particle size of the materials in themicronized composition can vary as well depending upon the applicationof the micronized composition. In one aspect, the micronized compositionhas particles that are less than 500 μm, less than 400 μm, less than 300μm, less than 200 μm, less than 100 μm, less than 50 μm, less than 25μm, less than 20 μm, less than 15 μm, less than 10 μm, less than 9 μm,less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, lessthan 4 μm, less than 3 μm, less than 2 μm, or from 2 μm, to 400 μm, from25 μm to 300 μm, from 25 μm to 200 μm, or from 25 μm to 150 μm. In oneaspect, the micronized composition has particles that have a diameterless than 150 μm, less than 100 μm, or less than 50 μm. In otheraspects, particles having a larger diameter (e.g. 150 μm to 350 μm) aredesirable. In all cases, the diameter of the particle is measured alongits longest axis.

In one embodiment, the size of the particles may be reduced tonano-range. As one skilled in the art would understand, nanoparticles ofplacental components may be desirable for the increased density and/orincreased release rate upon applying to the wound. Preferably, theparticle size of the micronized particles is from about 0.05 μm to about2 μm, from about 0.1 μm to about 1.0 μm, from about 0.2 μm to about 0.8μm, from about 0.3 μm to about 0.7 μm, or from about 0.4 μm to about 0.6μm. Alternatively, the particle size of the micronized particles is atleast 0.05 μm, at least 0.1 μm, at least 0.2 μm, at least 0.3 μm, atleast 0.4 μm, at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, atleast 0.8 μm, at least 0.9 μm, or at least 1 μm. Alternatively, theparticle size of the micronized particles is less than 1 μm, less than0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, less than0.1 μm, or less than 0.05 μm.

In other aspects, particles having a range of sizes and volumes arepreferred as such particles will impart differential release rates intothe wound. In one embodiment, particles having a range of mass to volumeratios can be prepared by either micronizing a mixture of a monolayergraft with multi-layer grafts (e.g., 2-10 layers) such that a range ofgraft sizes and volumes are provided. In another embodiment, particlesof varying surface area to volume ratios of the same tissue material canbe prepared by compressing the linear grafts into three-dimensionalshapes of varying sizes (round, elliptical, oblong, etc.). As surfacearea to volume ratio is increased, particle dissipation increases due tothe larger exposure area for endogenous enzymes, etc. This results in afaster rate of release of collagen types IV, V, and VII, cell-adhesionbio-active factors including fibronectin and laminins and othercomponents of the micronized particles. On the other hand, as thesurface area to volume ratio is decreased, particle dissipationdecreases due to the smaller exposure area for endogenous enzymes, etc.This results in a slower rate of release of collagen types IV, V, andVII, cell-adhesion bio-active factors including fibronectin and lamininsand other components of the micronized particles. In combination, theuse of a layer of micronized particles having different surface area tovolume ratios provides for a “time-release” mechanism whereby thebenefits of the micronized graft are both immediate and prolonged.

In one embodiment, the surface area to volume ratio (based on a spherehaving a range of diameters as described above) is between the range ofabout 0.06 μm to about 6×10⁴ μm, about 0.06 μm to about 6×10³ μm, about0.06 μm to about 6×10² μm, or about 0.6 μm to about 6×10² μm.

Preparation of Reinforced Tissue Grafts and Dehydration (Step 150)

After the placental tissue has been prepared, a reinforced tissue graftis produced by laminating one or more placental tissues on each side ofa biocompatible mesh. The biocompatible meshes useful herein generallyhave a plurality of pores. In one aspect, the biocompatible mesh can bemade from a sheet or film of material containing circular, elliptical,or other shaped pores. Pores may be formed in the sheet or film bypunching, drilling, milling, or other techniques known in the art. Thepores should be of sufficient size to allow self-adherence of theplacental tissue layers. In one aspect, the pore size can includediameters in the range from 200 to 4,000 microns and can be spaced from1,000 to 4,500 microns apart as measured from center of pore to centerof pore. The thickness of the sheet or film can range from 300 to 2,000microns. The pores in the biocompatible mesh may be chamfered, radiused,or other method commonly known to those skilled in the art in order toprevent the edges of the pores from cutting the placental tissue.

In other aspects, the mesh can be a textile mesh made by weaving,knitting, felting, or other textile methods known in the art usingfibers, wires, or yarns of a biocompatible material in order to create atextile mesh with pores. In one aspect, the pore size can range from 0.5mm to 3 mm in diameter. In another aspect, the thickness of the textilemesh can range from 300 to 2000 microns.

In one aspect, the biocompatible mesh can be made from non-resorbablematerials including but not limited to biocompatible metals such astitanium alloys, stainless steel, cobalt-chromium alloys, andnickel-titanium alloys. In another aspect, the layer of biocompatiblemesh can be made from non-resorbable polymeric materials, including butnot limited to, thermoplastic resins, polyethylenes, ultra-high weightmolecular weight polyethylene, high molecular weight polyolefins,uncoated monofilament polypropylene, polyether ether ketone,polyethylene terephthalate, polytetrafluoroethylene, expandedpolytetrafluoroethylene, nylon, any polymer or aliphatic hydrocarbonscontaining one or more double bonds, any other appropriate porousmaterials, or any other appropriate porous material that can be bent orotherwise formed into a shape.

In another aspect, the biocompatible mesh can be composed of a syntheticor biological resorbable polymeric material including but not limited topolyglycolic acid, poly-L-lactic acid (PLLA), poly-D,L-lactic acid(PDLA), trimethylenecarbonate (TMC), poly-ε-caprolactone,poly-P-dioxanone, copolymers of lactide and glycolide (PLGA),polyhydroxy-3-butyrate, collagen, hyaluronic acid, silk, biocellulose,other protein-based polymers, polysaccharides, poly(DTE carbonate),polyarylates, blends of PLLA, PLDA, or PLGA with TMC and othercombinations of these polymers.

The reinforced tissue grafts are generally a sandwich structure composedof one or more placental tissues laminated on each side of thebiocompatible mesh. The reinforced tissue grafts can have from 1 to 10placental tissues laminated on each side of the biocompatible mesh.Furthermore, the placental tissue can be any combination of tissues(e.g., amnion, chorion, Wharton's jelly, etc.). Finally, the placentaltissue can be optionally modified (e.g., removal of the epithelium cellsand/or intermediate layer) and/or cross-linked using the techniquesdescribed above.

In one aspect, the biocompatible mesh as described herein can be eitherstructurally homologous or heterologous in its configuration, wherein astructurally homologous biocompatible mesh is wholly composed fromplacental tissue, including, but not limited to, be amnion, chorion,Wharton's jelly and the like, and wherein a structurally heterologousbiocompatible mesh is composed from placental tissue that can be anycombination of placental tissues as described herein.

In other aspects, the biocompatible mesh as described herein can becoated with micronized placental tissue to provide a further amount ofplacental tissue for use as described herein. Micronized placentaltissue can be prepared by using instruments known in the art and asfurther described herein.

In another aspect, the micronized placental tissue can be injected intoa tissue graft or applied directly to a wound site as a jetted solutionusing a needle-free transdermal transport device. Jetting techniquesusing needle-free transdermal transport devices are known by those ofskill in the art. In certain aspects, jetting techniques may be used asa substitute method for applying micronized placental tissue.Alternatively, jetting techniques may be used to supplement additionalmicronized placental tissue to the tissue graft or wound site to enhancewound healing and other medical applications. In certain other aspects,the micronized placental tissue may be provided in any suitable mediumdepending on the jetting technique being used, including, but notlimited to, solutions, suspensions and powders.

In another aspect, the micronized placental tissue may be applied to thesurface of a membrane by first depositing the micronized placentaltissue onto a non-stick surface such as Teflon® and subsequentlythereafter contacting the interior surface of the membrane with thedeposited micronized placental tissue to absorb the micronized placentaltissue onto the interior surface of the membrane. In this aspect, thenon-stick surface can be sterilized according to conventional methods,prior to deposition of the micronized placental tissue. In certainaspects, the membrane or graft can be provided in a wet form tofacilitate adhesion of the micronized placental tissue to the membrane.In another aspect, a second membrane can be later applied onto the firstmembrane containing the micronized placental tissue to produce a tissuegraft.

In one aspect, the reinforced tissue graft includes:

a first membrane comprising a placental tissue having a first side and asecond side;

a biocompatible mesh having a first side and a second side, wherein thefirst side of the biocompatible mesh is adjacent to the second side ofthe first membrane; and

a second membrane comprising a placental tissue having a first side anda second side, wherein the first side of the second membrane is adjacentto the second side of the biocompatible mesh.

The reinforced tissue grafts can be configured in a number of differentconfigurations depending upon the application of the tissue graft. Inone aspect, the first membrane comprises amnion, chorion, or a laminatecomprising one or more layers of amnion with one or more layers ofchorion. In another aspect, the second membrane comprises amnion,chorion, or a laminate comprising one or more layers of amnion with oneor more layers of chorion. A number of different configurations of thereinforced tissue graft are depicted in FIG. 3 (A-G).

In one aspect, the first membrane comprises modified amnion wherein themodified amnion comprises a first side which is an exposed basementmembrane and a second side, and wherein the second side of the modifiedamnion is adjacent to the first side of the biocompatible mesh. Inanother aspect, the first membrane comprises modified amnion wherein themodified amnion comprises a first side which is an exposed basementmembrane and a second side which is an exposed fibroblast layercomprising fibroblast cells, and wherein the second side of the modifiedamnion is adjacent to the first side of the biocompatible mesh.

In another aspect, the second membrane comprises an amnion/chorionlaminate, wherein the chorion is adjacent to the second side of thebiocompatible mesh.

In a further aspect, the second membrane comprises an amnion/chorionlaminate, wherein the chorion is adjacent to the second side of thebiocompatible mesh, the amnion comprises an epithelium layer and anintermediate layer, and the chorion is adjacent to the intermediatelayer.

In a further aspect, the second membrane comprises an amnion/chorionlaminate, wherein the chorion is adjacent to the second side of thebiocompatible mesh, the amnion comprises a modified amnion comprising anexposed basement membrane and an intermediate layer, and the chorion isadjacent to the intermediate layer.

In a further aspect, the second membrane comprises an amnion/chorionlaminate, wherein the chorion is adjacent to the second side of thebiocompatible mesh, the amnion comprises a modified amnion comprising anexposed basement membrane and an exposed fibroblast layer comprisingfibroblast cells, and the chorion is adjacent to the exposed fibroblastlayer.

In one aspect, the reinforced tissue graft is composed of a layer ofamnion (i.e., first membrane) where the epithelium layer has beensubstantially removed in order to expose the basement layer to hostcells, a biocompatible mesh, and a layer of amnion (i.e., secondmembrane) (with or without the epithelia cells) (FIG. 3A). Here, theexposed basement layer is not adjacent to the biocompatible mesh.

In another aspect, the reinforced tissue graft is composed of a layer ofamnion (with layer of epithelial cells), a layer of chorion, a layer ofbiocompatible mesh, and a layer of amnion (with or without theepithelial cells). In this aspect, the layer of amnion and chorioncollectively are the first membrane (FIG. 3B). Here, the exposedbasement layer or the epithelial layer of the amnion is not adjacent tothe biocompatible mesh.

In another aspect, the reinforced tissue graft is composed of layer ofamnion where the epithelium layer has been substantially removed inorder to expose the basement layer to host cells, a layer of chorion, asecond layer of amnion (with layer of epithelia cells), a second layerof chorion, a layer of biocompatible mesh, a layer of amnion (with layerof epithelia cells), and a layer of chorion (FIG. 3F).

In a further aspect, the reinforced tissue graft is composed of a layerof amnion where the epithelium layer has been substantially removed inorder to expose the basement layer to host cells, a layer of chorion, alayer of biocompatible mesh, and a layer of Wharton's jelly (FIG. 3G).Here, the exposed basement layer is not adjacent to the biocompatiblemesh.

In another aspect, the reinforced tissue graft is composed of a layer ofWharton's jelly where the outer layer of epithelium is removed, a layerof biocompatible mesh, a layer of chorion, and a layer of amnion wheresubstantially all of the epithelium cells are removed to expose thebasement membrane (FIG. 3G). Here, the side of the Wharton's jelly wherethe epithelial cells have been removed are not adjacent to thebiocompatible mesh. Additionally, the exposed basement membrane of theamnion is not adjacent to the chorion.

In certain aspects, a bioactive agent can be added to the placentaltissue prior to and/or after lamination and production of the reinforcedtissue graft. Examples of bioactive agents include, but are not limitedto, naturally occurring growth factors sourced from plateletconcentrates, either using autologous blood collection and separationproducts, or platelet concentrates sourced from expired banked blood;bone marrow aspirate; stem cells derived from concentrated humanplacental cord blood stem cells, concentrated amniotic fluid stem cellsor stem cells grown in a bioreactor; or antibiotics. Upon application ofthe reinforced tissue graft with bioactive agent to the region ofinterest, the bioactive agent is delivered to the region over time.Thus, the reinforced tissue grafts described herein are useful asdelivery devices of bioactive agents and other pharmaceutical agentswhen administered to a subject.

Release profiles can be modified based on, among other things, thedegree of cross-linking in the placental tissue grafts used to preparethe reinforced tissue graft. In certain aspects, the tissue graftsdescribed herein are useful in wound healing applications where it isdesirable to keep a bioactive agent localized in the wound so that thewound heals quicker. Additionally, if the bioactive agent is toxic whenreleased systemically throughout the subject, the reinforced tissuegrafts described herein can provide an effective, impermeable barrierthat prevents the bioactive agent from migrating from the wound. Forexample, the placental tissue can be sufficiently cross-linked in orderto prevent a bioactive agent such as INFUSE® from leaching out of thereinforced tissue graft. Here, the cross-linked placental tissue in thereinforced tissue graft acts as a barrier.

The preparation of the reinforced tissue grafts generally involves thesequential layering of placental tissue on the biocompatible mesh. Inone aspect, one or more placental tissues (i.e., the first membrane) canbe placed on the surface of a drying fixture. Next, the biocompatiblemesh is applied to the first membrane, with the subsequent layering ofone or more additional placental tissues (i.e., the second membrane) onthe biocompatible mesh.

In certain aspects, adhesives such as, for example fibrin glue andhydrogels, can be used to adhere the placental tissues together as wellas to the biocompatible mesh. Fibrin glue is prepared from pooled bloodand has the potential to transmit disease. At this time, the applicationof fibrin glue to seal dural tears constitutes off label use. Synthetichydrogels such as the DuraSeal Spine Sealant System (Confluent SurgicalInc., Waltham, Mass.) consist of two components (polyethylene glycolester and trilysine amine) and a delivery system which polymerize at thedefect site to form a seal. As the hydrogel swells to up to 50% in sizeduring polymerization, neural compression may occur.

The drying fixture is preferably sized to be large enough to receive theplacental tissue, fully, in laid out, flat fashion. In one aspect, thedrying fixture is made of Teflon or of Delrin, which is the brand namefor an acetal resin engineering plastic invented and sold by DuPont andwhich is also available commercially from Werner Machine, Inc. inMarietta, Ga. Any other suitable material that is heat and cutresistant, capable of being formed into an appropriate shape to receivewet tissue can also be used for the drying fixture.

In one aspect, similar to that shown in FIG. 2, the receiving surface ofthe drying fixture 500 has grooves 505 that define the product spaces510, which are the desired outer contours of the tissue after it is cutand of a size and shape that is desired for the applicable surgicalprocedure in which the tissue will be used. For example, the dryingfixture can be laid out so that the grooves are in a grid arrangement.The grids on a single drying fixture may be the same uniform size or mayinclude multiple sizes that are designed for different surgicalapplications. Nevertheless, any size and shape arrangement can be usedfor the drying fixture, as will be appreciated by those skilled in theart. In another embodiment, instead of having grooves to define theproduct spaces, the drying fixture has raised ridges or blades.

Within the “empty” space between the grooves or ridges, the dryingfixture can include a slightly raised or indented texture in the form ofan indicia 520 (e.g., a text, logo, name, or similar design). Here, theindicia can be seen by the naked eye (with or without corrective lenses)and not by magnification techniques. This textured text, logo, name, ordesign can be customized. When dried, the tissue will mold itself aroundthe raised texture or into the indented texture—essentially providing alabel within the tissue itself. Preferably, the texture/label can beread or viewed on the tissue graft in only one orientation so that,after drying and cutting, an end user (typically, a clinician) of thedried tissue will be able to tell the stromal side from the basementside of the dried tissue. The reason this is desired is because, duringa surgical procedure, it is desirable to place the allograft in place,with amnion basement side down or adjacent the native tissue of thepatient receiving the allograft. FIG. 2 illustrates a variety of marks,logos, and text 520 that can be included within the empty spaces 510 ofthe drying fixture 500. Typically, a single drying fixture will includethe same design or text within all of the empty spaces; however, FIG. 2shows, for illustrative purposes, a wide variety of designs that can beincluded on such drying fixtures to emboss each graft.

In one aspect, after the reinforced tissue graft has been produced andprior to dehydration, pressure can be applied to the tissue graft suchthat the first and second membrane are pressed into the pores of thebiocompatible mesh and come into contact with one another. In oneaspect, a dry roller is rolled over the reinforced tissue graft. Inanother aspect, the biocompatible mesh is pressed into the placentalallograft using a mechanical press that only comes into contact with thebiocompatible mesh. In this aspect, the biocompatible mesh is pressedinto the first membrane, where the second membrane is subsequentlyapplied on the biocompatible mesh. In another aspect, a wetted platewith raised knobs can be placed on the reinforced tissue graft andpressed down such that the layers of placental tissue come into contactwith one another.

Once the reinforced tissue graft is produced, the reinforced tissuegraft is dehydrated. In one aspect, the drying fixture with thereinforced tissue graft is placed in a freeze-dryer. The use of thefreeze-dryer to dehydrate the tissue grafts can be more efficient andthorough compared to other techniques such as thermal dehydration. Ingeneral, it is desirable to avoid ice crystal formation in the placentaltissue grafts as this may damage the extracellular matrix in the tissuegraft. By chemically dehydrating the placental tissue prior tofreeze-drying, this problem can be avoided.

In another aspect, the dehydration step involves applying heat to thetissue graft. In one aspect, the drying fixture with the reinforcedtissue graft is placed in a sterile Tyvex (or similar, breathable,heat-resistant, and sealable material) dehydration bag and sealed. Thebreathable dehydration bag prevents the tissue from drying too quickly.If multiple drying fixtures are being processed simultaneously, eachdrying fixture is either placed in its own Tyvex bag or, alternatively,placed into a suitable mounting frame that is designed to hold multipledrying frames thereon and the entire frame is then placed into a larger,single sterile Tyvex dehydration bag and sealed.

The Tyvex dehydration bag containing the one or more drying fixtures isthen placed into a non-vacuum oven or incubator that has been preheatedto approximately 35 to 50 degrees Celcius. The Tyvex bag remains in theoven for between 30 to 120 minutes. In one aspect, the heating step canbe performed at 45 minutes at a temperature of approximately 45 degreesCelcius to dry the tissue sufficiently but without over-drying orburning the tissue graft. The specific temperature and time for anyspecific oven will need to be calibrated and adjusted based on otherfactors including altitude, size of the oven, accuracy of the oventemperature, material used for the drying fixture, number of dryingfixtures being dried simultaneously, whether a single or multiple framesof drying fixtures are dried simultaneously, and the like.

In certain aspects, once the reinforced tissue graft has been applied tothe drying fixture, a drying frame can be applied over the graft. Thisfeature is depicted in FIG. 4, where the drying rack 82 is placed on topof drying fixture 80. The drying frame holds the graft in place.Additionally, the drying frame allows the entire sheet of tissue graftto dry completely without lifting, which results in increased yields.

In another aspect, the reinforced tissue graft is dehydrated by chemicaldehydration followed by freeze-drying. In one aspect, the chemicaldehydration step is performed by contacting the placental tissueindependently or as a laminate with a polar organic solvent for asufficient time and amount in order to substantially (i.e., greater than90%, greater than 95%, or greater than 99%) or completely removeresidual water present in the placental tissue (i.e., dehydrate thetissue). The solvent can be protic or aprotic. Examples of polar organicsolvents useful herein include, but are not limited to, alcohols,ketones, ethers, aldehydes, or any combination thereof. Specific,non-limiting examples include DMSO, acetone, tetrahydrofuran, ethanol,isopropanol, or any combination thereof. In one aspect, the placentaltissue is contacted with a polar organic solvent at room temperature. Noadditional steps are required, and the tissue can be freeze-drieddirectly as discussed below.

After chemical dehydration, the reinforced tissue graft is freeze-driedin order to remove any residual water and polar organic solvent. In oneaspect, the reinforced tissue graft can be laid on a suitable dryingfixture prior to freeze-drying.

In another aspect, the placental tissue grafts described herein can bedehydrated using an innovative dehydration device which enhances therate and uniformity of the dehydration process. In one embodiment, thedrying time can be accelerated by up to 40% in one configuration of thedehydration device in comparison to conventional drying ovens. Incertain aspects, the placental tissue graft is placed onto a dryingfixture described herein and the drying fixture with tissue graft isinserted into the dehydration device for performing the dehydrationprocess. In other aspects, multiple placental tissue grafts can beplaced onto the drying fixture to dry more than one placental tissuegrafts in the dehydration device at the same time. Although thedehydration device is useful in dehydrating the tissue grafts describedherein, they can be used for dehydrating objects other than placentaltissue.

FIGS. 8-11 show an innovative dehydration device 900 according to anexample embodiment that is well-suited for use in the herein-describeddehydration processes. The dehydration device 900 includes a dryinghousing 902, and inflow plenum 904, and outflow plenum 906, anair-moving assembly 908, an air-heating assembly 910, and a controlsystem 912.

The drying housing 902 defines a drying chamber into which the placentaltissue (e.g., ton a drying fixture) is placed for drying during thedehydration process. In typical embodiments, the drying housing 902 (andthus the drying chamber it defines) is formed by six generally planarwalls arranged together in a generally rectanguloid shape. In otherembodiments, the drying housing 902, and/or the drying chamber itdefines, has a different regular or irregular shape such as spherical orellipsoidal. In the depicted embodiment, the drying housing 902 isformed by top and bottom opposing walls 914 and 916, first and secondopposing sidewalls 918 and 920, and first and second opposing endwalls922 and 924. The drying housing 902 includes a doorway opening 926 and adoor 928 (e.g. hingedly coupled to the housing and including apull-knob) in at least one of the walls (e.g., sidewall 918) forinserting the placental tissue on a fixture for dehydration and thenremoving the dried tissue. (FIG. 8 shows the door 928 in a closedposition and FIG. 9 shows it in an opened position.) The walls of thehousing 902 are typically made of a material selected for rigidity,strength, and heat-resistance, for example an acrylic (e.g., PLEXIGLAS),glass, ceramic, or other polymeric material.

At least two of the walls of the housing 902 each define at least onerespective aperture through which air can flow. In the depictedembodiment, for example, the top and bottom opposing walls 914 and 916have an array of inflow and outflow apertures 930 and 932, respectively,formed in them. In such embodiments, the placental tissue graft (e.g.,on a fixture) is placed into the drying chamber supported by the bottomwall 916 and typically at least partially covering at least one of theoutflow apertures 932. The size, shape, and position of the apertures930 and 932 are selected based on the range of operating parameters(volumetric flow rate, flow pattern, temperature, pressure,time/duration, etc. of the air flowing through the housing 902) of thedevice 900 as may be desired for drying the placental tissue. Thus, theapertures 930 and 932 can be circular, aligned with correspondingapertures in the opposing wall, arranged in segmented rows and/orcolumns, and arranged uniformly (for a generally uniform temperature anddrying effect across the chamber), as depicted. In other embodiments,the apertures have a non-circular shape (e.g., polygonal or elliptical),have differing sizes (e.g., interspersed larger and smaller apertures,or differing inflow and outflow aperture sizes), and/or are formed in anirregular and/or non-aligning pattern. And in yet other embodiments, theapertures are formed in only one of the walls, more than two of thewalls, or the opposing sidewalls 918 and 920 (instead of or in additionto the opposing top and bottom walls 914 and 916), and/or the inflowplenum 904 can be eliminated and piping coupled between the air-movingassembly 908 and an inflow one of the walls (e.g., top wall 914).

The inflow plenum 904 and the outflow plenum 906 are positioned incommunication with the inflow apertures 930 and the outflow apertures932, respectively. The plenums 904 and 906 help generate an evendistribution of the pressure, flow, and temperature of the air flowingthrough the drying housing 902. In the depicted embodiment, the inflowplenum 904 is formed by first vertically upward extensions of theopposing sidewalls 918 and 920 and the opposing endwalls 922 and 924together with the housing top wall 914 and an opposing inflow-plenum topwall 934. And the outflow plenum 906 is formed by second verticallydownward extensions of the opposing sidewalls 918 and 920 and theopposing endwalls 922 and 924 together with the housing bottom wall 916and an opposing outflow-plenum bottom wall 936. In other embodiments,the plenums 904 and 906 are eliminated and the air-moving assembly 908is piped directly to the drying housing 902.

The inflow plenum 904 and the outflow plenum 906 include at least oneinflow port 938 and outflow port 940, respectively. In the depictedembodiment, the inflow port 938 is defined by a generally rectangularopening formed in the sidewall 920 at an upper portion thereof and at afirst/distal portion thereof, and the outflow port 940 is defined by agenerally rectangular gap in the same sidewall (i.e., an absence of thesecond extension of the wall) but at a lower portion thereof and at asecond/proximal portion thereof. In this way, the air flows laterallyinto the inflow plenum 904 at the first/distal and upper portion of thedehydration device 900 and then distributes proximally within the inflowplenum. Then the air flows down through the inflow apertures 930, downthrough and across the drying chamber, down through the outflowapertures 932, down into the outflow plenum 906, and laterally out atthe second/proximal and lower portion of the device 900. The plenums 904and 906 provide for generally evenly distributed airflow across thetissue even though the air enters the inflow plenum at the first/distalportion of the dehydration device 900 and exits the outflow plenum atthe second/proximal portion (while flowing from top to bottom throughthe drying chamber). Alternatively, the inflow and outflow ports 938 and940 can be positioned to provide airflow from bottom to top (and/or fromside to side) through the drying chamber, and/or they can have otherregular or irregular shapes such as circular.

The air-moving assembly 908 can be of a commercially available type foruse in sterile/clean-air environments such as medical laboratories.Typically, the air-moving assembly 908 includes a blower 942 and afilter 944. The blower 942 can be of a conventional type, for exampleincluding an electric motor and a fan enclosed within a housing. And thefilter 944 can be of a conventional type, for example a cylindrical HEPAair filter with an internal bore. Typically, such filter 944 mounts toand extends from the blower 942, and air flows axially through theinternal bore and radially outward through the filter media.

The dehydration device 900 can be configured in a closed airflow loop(to re-circulate the air) or in an open loop (to provide fresh intakeair). In closed-loop designs, an air outlet surface 946 of the filter944 is in sealed communication with the inflow port 938 of the inflowplenum 904, and an air intake 948 of the blower 942 is in sealedcommunication with the outflow port 940 of the outflow plenum 906. Inthe depicted embodiment, for example, the air outlet surface 946 of thefilter 944 is enclosed in a first/distal delivery chamber formed bylateral extensions of the plenum top and bottom walls 934 and 936, alateral extension of the first/distal endwall 922 and an opposingsecond/proximal delivery-chamber endwall 950, and the second sidewall920 and an opposing delivery-chamber sidewall 952. And the air intake948 of the blower 942 is sealed communication with a second/proximalreturn chamber formed by lateral extensions of the plenum top and bottomwalls 934 and 936, a lateral extension of the second/proximal endwall924 and an opposing first/distal return-chamber endwall 954 (having anreturn opening in sealed communication with the blower air intake), andthe second sidewall 920 and an opposing return-chamber sidewall 956. Asidewall section can be provided to enclose the blower 942 or this canbe left out to allow ambient air exposure to prevent the blower fromoverheating. In the depicted embodiments, the result is that the outerwalls of the dehydration device 900 form a rectanguloid structure. Inother embodiments, the air outlet surface 946 of the filter 944 is pipedto the inflow port 938 of the inflow plenum 904 and the air intake 948of the blower 942 is piped to the outflow port 940 of the outflow plenum906.

The air-heating assembly 910 includes at least one heating element 958,which can be of a conventional type such as a commercially availableelectric-resistance heating element. The heating element 958 istypically positioned adjacent the air intake 948 of the blower 942, forexample mounted on a bracket within the return chamber, as depicted.

The control system 912 includes conventional controls for controllingthe operating parameters (airflow rate, pressure, temperature,time/duration, etc.) of the dehydration device 900. Such conventionalcontrols typically include a main power switch 960 that is wired toprovide power to a variable resistance device 962 and a control unit964. The main power switch 960 is wired to a power source such asconventional 120/240 line voltage. The variable resistance device 962(e.g., a rheostat) is wired (for power and control) to the heatingelement 958 (e.g., via the control unit 964) for temperature control. Atleast one heat sensor 966 is positioned in the return chamber and wiredto the control unit 964 to provide an input for use in temperaturecontrol. And the control unit 964 is wired (for power and control) tothe blower 942 for controlling the volume flow rate (and thus also thepressure) and the time/duration of the dehydration cycle. In addition,typical embodiments such as that depicted include a pressure sensor 968in (or at least exposed to) the drying chamber, a pressure gauge display970 (e.g., mounted to the drying housing 902), and a fluid connection972 (e.g., tubing) interconnecting the two parts.

Cutting & Packaging (Step 160)

Once the reinforced tissue graft has been adequately dehydrated, thetissue graft is then ready to be cut into specific product sizes andappropriately packaged for storage, terminal sterilization, and latersurgical use. In one aspect, the Tyvek bag containing the dehydratedtissue is placed back into the sterile/controlled environment. Thenumber of grafts to be produced is estimated based on the size and shapeof the tissue on the drying fixture(s). An appropriate number ofpouches, one for each tissue graft, is also introduced into thesterile/controlled environment. The drying fixture(s) are then removedfrom the Tyvek bag.

If the drying fixture has grooves, then the following exemplaryprocedure can be used for cutting the tissue graft into product sizes.If the drying fixture is configured in a grid pattern, a #20 or similarstraight or rolling blade is used to cut along each groove line inparallel. Next, all lines in the perpendicular direction are cut.Alternatively, if the drying fixture has raised edges or blades, thenthe following procedure can be used for cutting the tissue graft intoproduct sizes. A sterile roller is used to roll across the dryingfixture. Sufficient pressure must be applied so that the dehydratedtissue graft is cut along all of the raised blades or edges of thedrying fixture.

After cutting, each tissue graft is placed in a respective “inner”pouch. The inner pouch, which preferably has a clear side and an opaqueside, should be oriented clear side facing up. The tissue graft isplaced in the “inner” pouch so that the texture in the form of text,logo, name, or similar design is facing out through the clear side ofthe inner pouch and is visible outside of the inner pouch. This processis repeated for each separate tissue graft.

Each tissue graft is then given a final inspection to confirm that thereare no tears or holes, that the product size (as cut) is withinapproximately 1 millimeter (plus or minus) of the specified length andwidth size and within approximately 250 microns (plus or minus) thickfor that particular graft, that there are no noticeable blemishes ordiscoloration of the tissue graft, and that the textured logo or wordingis readable and viewable through the “inner” pouch.

To the extent possible, oxygen is removed from the inner pouch before itis sealed. The inner pouch can be sealed in any suitable manner;however, a heat seal has shown to be effective. In one aspect, afterpackaging, the product is terminally sterilized by radiation, usinggamma or electron beam sterilization with a target dose of, for example,17.5 kGy. Next, each inner pouch is separately packaged in an “outer”pouch for further protection, storage, and shipment.

It should be noted that none of the steps described above involvefreezing the tissue graft to kill unwanted cells, to decontaminate thetissue graft, or otherwise to preserve the tissue graft. The dehydratedtissue grafts described herein are designed to be stored and shipped atroom or ambient temperature without need for refrigeration or freezing.

Product Release (Step 170)

Before the reinforced tissue graft is ready for shipment and release tothe end user, all documentation related to the manufacture, recovery anddonor eligibility are reviewed and deemed acceptable by the qualityassurance department and the medical director.

Appropriate labeling and chain of custody is observed throughout all ofthe above processes, in accordance with accepted industry standards andpractice. Appropriate clean room and sterile working conditions aremaintained and used, to the extent possible, throughout the aboveprocesses.

II. Applications of Reinforced Tissue Grafts

Due to the enhanced adhesive nature structural features of thereinforced tissue grafts described herein, the grafts can be used innumerous medical applications involving wound healing in a subject. Inone aspect, when the placental tissue is cross-linked, the cross-linkinggroups covalently attached to the tissue graft can facilitate thenon-enzymatic cross-linking of proteins within the graft such as, forexample, collagen, and other proteins present in a biological tissue. Inone aspect, cross-linked tissue grafts described herein can cross-link(i.e., form a covalent bond) with dura matter. In other aspects, thetissue grafts described herein can adhere to tendons, ligaments, muscle,and other body tissue. The tissue grafts described herein are useful inthe reinforcement and sealing of tears as well as the prevention orreduction of scar formation after surgery in addition to otherpost-surgical complications. Additionally, due to the enhanced adhesiveproperties of the tissue graft, the grafts are ready for application tothe surgical site without the need for sutures.

In one aspect, the grafts described herein are useful in enhancing orimproving wound healing. The types of wounds that present themselves tophysicians on a daily bases are diverse. Acute wounds are caused bysurgical intervention, trauma and burns. Chronic wounds are wounds thatare delayed in closing compared to healing in an otherwise healthyindividual. Examples of chronic wound types plaguing patients includediabetic foot ulcers, venous leg ulcers, pressure ulcers, arterialulcers, and surgical wounds that become infected.

The physician's goal when treating traumatic wounds is to heal the woundwhile allowing the patient to retain natural function in the area of thewound with minimal scaring and infection. If a wound becomes infected,it can lead to a loss of limb or life. For the most part, physiciansheal these patients without incident. However, physicians dealing withchronic wounds are mainly concerned with closing the wound as quickly aspossible to minimize the risk of an infection that could lead to loss oflimb or life. Chronic wounds are wounds on patients that havecomorbidities that complicate or delay the healing cascade. In oneaspect, the grafts described herein can function as a tissueregeneration template that delivers essential wound healing factors,extracellular matrix proteins and inflammatory mediators to help reduceinflammation, enhance healing, and reduces scar tissue formation. Inthis aspect, the micronized placental compositions described herein areused in treating wounds amenable to negative pressure technology,including burns and ulcers, such as chronic ulcers, diabetic ulcers,decubitus ulcers and the like.

In another aspect, the micronized placental tissue is used inconjunction with conventional treatments, including, but not limited to,negative pressure therapy, and may also be used in combination withmatrices or scaffolds comprised of biocompatible materials, such ascollagen, hyaluronic acid, gelatin or combinations thereof.

In another aspect, the tissue grafts described herein are useful foraddressing or alleviating complications to the spine and surroundingregions that occur after surgery. Acute and chronic spinal injuries andpain can be attributed to trauma and/or degenerative changes in thespinal column. For the degenerative patient, there is usually aprogression of possible surgeries depending on the patient's symptomsand disease state. The first surgical option when conservative therapyhas failed is a laminectomy or micro-discectomy. These minimallyinvasive procedures are intended to relieve the pain generator orstenosis of the spinal canal. If there is progression of the disease,then other surgeries may be necessary including, but not limited to, aspinal fusion. Spinal fusions may be achieved through severalapproaches: anterior (from the front through the abdomen), posterior(from the back), or lateral (through the side). Each approach hasadvantages and disadvantages. The goal is typically to remove the spinaldisc, restore disc height and fuse the two spinal vertebrae together tolimit motion and further degradation. There are also surgical optionsfor the surgeon and patient to replace the spinal disc with anartificial disc. Spine trauma is typically treated by fusing the spinelevels or if a vertebrae is crushed, the surgeon may choose to do acorpectomy and fusing across the levels that were affected.

In one aspect, the tissue grafts described herein are useful inpreventing or reducing scar formation on the spine or near the spine andsealing dural tears. Scar formation at or near the spine after surgerycan be very debilitating and possibly require subsequent operations toaddress the symptoms as discussed above. The term “anti-adhesion” isalso used in the art to refer to the prevention of scar tissue at ornear the spine. In other aspects, the tissue grafts described herein canbe used as a protective barrier, where the graft protects the spinaldura from post-surgical trauma from the surrounding surgical site. Forexample, the grafts can prevent damage to the spinal dura caused bysharp edges from newly cut bone such as vertebrae. In other aspects, thetissue grafts can be used for anterior lumbar interbody fusion,posterior lumbar interbody fusion trans-lumbar interbody fusion,anterior cervical discectomy and fusion, micro discectomy, spinal durarepair, and as a dura sealant to prevent CSF leakage.

Depending upon the surgical procedure, the tissue graft can be applieddirectly to the spinal dura, the surrounding region of the spine toinclude nerve roots, or a combination thereof. Due to the uniquestructure of vertebrae, the tissue graft can be cut into any shape ordimension so that it can be placed and affixed at the appropriateposition in the subject. For example, when the tissue graft is used forbi-lateral coverage, membranes in the shape of a rectangle allow thetissue graft to fit around the posterior spinal process, which minimizeslateral movement. In addition to minimizing lateral movement, the tissuegraft can also provide proximal and distal barrier coverage where thespinal lamina has been removed for exposure to the affected area. In oneaspect, to ensure proper placement, the graft can be embossed on theexposed basement membrane of the graft to ensure proper placement of thegraft in the subject. In particular, proper graft placement will ensurethat the basement membrane of the graft is in direct contact with thespinal dura or surrounding region. For example, proper membraneplacement and orientation is important when applying the material inspinal applications where a posterior or anterior approach is utilized.

The grafts are useful in preventing or reducing scar formation that canresult from a variety of surgical procedures associated with the spine.The grafts can be used after any procedure in the neck, mid-back, orlower back. Depending upon the application, the epithelium of the amnionmembrane can be substantially removed. For example, in posteriorprocedures such as a laminectomy or discectomy, the epithelium layer issubstantially removed. Removal of the epithelial cell layer exposes theamnion's basement membrane layer, which increases cell signalingcharacteristics. This up regulation response enhances cellular migrationand expression of anti-inflammatory proteins, which inhibits fibrosis.The spinal dura is typically left unprotected following posteriorprocedures. Thus, the grafts described herein provide an unmet need inthese procedures.

In other aspects, the epithelial cell layer is not removed. For example,in anterior procedures or modified anterior procedures such as AnteriorLumbar Interbody Fusion (ALIF) and Transforaminal Interbody Fusion(TLIF), the amnion epithelium layer is not removed and remains intact.In these aspects, the grafts provide additional protection to thevertebral surgical site by maintaining separation from the peritoneum,larger vessels, and abdominal musculature. The membrane serves as areduced friction anatomical barrier against adhesions and scaring. Forexample, the grafts can prevent scar tissue binding major blood vesselsto the spine. This is a common problem with post-spinal surgery, whichrequires a second surgical procedure to address this.

In another aspect, the tissue grafts are useful in dental applications.For example, the grafts can be used around dental implants or in thetreatment of advanced gingival recession defect. In another aspect, thegrafts can be used in guided tissue regeneration.

In other aspects, the grafts described herein can be used in orthopedicapplications (i.e., sports medicine). Sports medicine includes therepair and reconstruction of various soft-tissue injuries in or aroundjoints caused by traumas, or chronic conditions brought about byrepeated motion, in active individuals and athletes. For example, sportsmedicine includes the treatment of a variety of different injuriesassociated with, but not limited to, shoulders, elbows, feet, ankleshand and wrists.

The main types of injuries include tendon and ligament sprains andruptures in the various joints, with the most common being ACL in theknee and rotator cuff in the shoulder. Non-tendon and ligamentprocedures include repair of torn knee meniscus and repair of kneecartilage which if left un-treated can lead to osteoarthritis of thejoint. Non-surgical options also include injections of anti-inflammatorydrugs to inflamed tendons (such as “tennis elbow”), injection oflubricants into joints (such as hyaluronic acid into the knee), as wellas physiotherapy and bracing.

In one aspect, the tissue grafts described herein can be used to wraptendon repairs to prevent scar formation on the healing tendon. They canalso provide a protective, enclosed environment for the repair toprogress successfully. The tissue grafts can be used as an off-the-shelftendon and ligament to replace the need to purchase an allograft orperform tendon or ligament transfer.

In other aspects, the tissue grafts described herein can be used in thereinforcement of rotator cuffs. Some rotator cuff tears are large enoughthat they require a reinforcement matrix to support the repair due tolack of viable native tissue. The tissue grafts described herein can beused as a matrix to reinforce a repair. In one aspect, the tissue graftsdescribed herein can be used to repair knee cartilage. For example, thetissue grafts can be used as a barrier to hold cell culturedchondrocytes or other pro-cartilage regeneration matrix inside achondral defect. In this aspect, the tissue graft would be utilized as aflap to close the defect and hold the matrix in place.

In one aspect, the tissue grafts can be used to repair peripheralnerves. The tissue graft can be used as a wrap on nerve repairs toprevent scar formation onto the healing nerve. The tissue graft can alsoprovide a protective enclosed environment for the repair to progresssuccessfully. In other aspects, the tissue grafts can be manufacturedinto a nerve regeneration tube to guide nerve growth in a protectiveenvironment where the nerve ends cannot be re-approximated. Here, nervescan re-attach up to a certain distance if the ends are allowed to meetfreely without other soft tissue interfering. In another aspect, thetissue graft can be used to wrap nerve bundles after prostatectomyprocedures. These nerves are responsible for erectile function andpossible continence. The tissue grafts can be laid on the nerves to keepthem from scarring and possibly damaging the nerves.

In other aspects, the tissue grafts described herein can be used inother orthopedic applications such as aid in the repair of periostium;help repair ruptured/damaged bursa; help secure void filling materialduring bone repair; or in applications involving a subject's extremities(e.g., anti-adhesion barrier for small bone fixation, anti-adhesionbarrier where metal plating or hardware is used, or help repairruptured/damaged bursa).

In another aspect, the tissue grafts can be used in obstetrics andgynecological (OB/GYN) surgical procedures involving the treatment ofdiseases that may be related to the fertility of the female, pain causedby the reproductive system or cancer in the reproductive system. Theseprocedures include the removal of uterine fibroids (myomectomy), removalof ovarian cysts, tubal ligations, endometriosis treatments, removal ofsome cancerous or non-cancerous tumors, and vaginal slings. Theseprocedures may be completed through a transvaginal, abdominal orlaproscopical approach.

The tissue grafts can be used as a patch to reduce the amount of scartissue in the reproductive system after a surgical procedure. Scartissue is another form of fibrous tissue and may also contribute tofertility problems. The ability to minimize the amount of scar on theovaries, or within the fallopian tubes may help with post-operativefertility and even pain. In another aspect, the tissue grafts can beused to reline the uterine wall after severe endometriosis treatmentsand increase the patient's ability to conceive. In a further aspect, thetissue graft can be used as an anti-adhesion barrier after removal ofovarian cyst or aid in the repair of vaginal wall erosion.

In other aspects, the tissue grafts can be used in cardiac applications.Angina is severe chest pain due to ischemia (a lack of blood, thus alack of oxygen supply) of the heart muscle, generally due to obstructionor spasm of the coronary arteries (the heart's blood vessels). Coronaryartery disease, the main cause of angina, is due to atherosclerosis ofthe cardiac arteries. Various open cardiac and vascular surgeryprocedures to remove atherosclerotic clots require the repair,reconstruction and closure of the vessel, and the support of aregenerative tissue patch to close and patch the surgical defect. Heartby-pass grafts and heart defect reconstruction (as part of an open-heartsurgical procedure) also can benefit from a patch or graft to provide abuttress to soft-tissue weakness, tissue replacement if there is a lackof suitable tissue, and also the potential to reduce adhesions to theheart itself. The tissue grafts described herein can be used as a patchto support the repair of vascular and cardiac defects caused byoperations and complications such as carotid artery repair, coronaryartery bypass grafting, congenital heart disease, heart valve repair,and vascular repair (i.e. peripheral vessels). In other aspects, thereinforced tissue graft can be configured into a stent (FIG. 7).

The tissue grafts described herein can be used in general surgeryprocedures. For example, general surgical procedures include proceduresrelated to the abdominal cavity. These include the intestines, stomach,colon, liver, gallbladder, appendix, bile ducts and thyroid glands.Procedures may include hernias, polypectomy, cancer removal, surgicaltreatment of Crohn's and ulcerative colitis. These procedures may bedone open or laparoscopically. In other aspects, the tissue grafts canbe used to facilitate closure of anastomosis, an anti-adhesion barrierfor anastomosis, or an anti-adhesion barrier for hernia repair.

In other aspects, the tissue grafts can be used in ENT procedures.Tympanoplasty is performed for the reconstruction of the eardrum(tympanic membrane) and/or the small bones of the middle ear. There areseveral options for treating a perforated eardrum. If the perforation isfrom recent trauma, many ear, nose and throat specialists will elect towatch and see if it heals on its own. If this does not occur or frequentre-perforation occurs in the same area, surgery may be considered.Tympanoplasty can be performed through the ear canal or through anincision behind the ear. Here, the surgeon harvests a graft from thetissues under the skin around the ear and uses it to reconstruct theeardrum. The tissue grafts described herein can be used to prevent theadditional trauma associated with harvesting the patients' own tissueand save time in surgery. In other aspects, the tissue grafts can beused as a wound covering after adenoidectomy, a wound cover aftertonsillectomy, or facilitate repair of the Sniderian membrane.

In other aspects, the tissue grafts described herein can be used inplastic surgery procedures. Scar revision is surgery to improve orreduce the appearance of scars. It also restores function and correctsskin changes (disfigurement) caused by an injury, wound, or previoussurgery. Scar tissue forms as skin heals after an injury or surgery. Theamount of scarring may be determined by the wound size, depth, andlocation; the person's age; heredity; and skin characteristics includingskin color (pigmentation). Surgery involves excision of the scar andcareful closure of the defect. In one aspect, the tissue graftsdescribed herein can be used as a patch to aid in the healing andprevention of scars; and keloid or cancer revision/removal where carefulapproximation of soft-tissue edges is not achievable and scar tissue canresult. Additionally, the anti-inflammatory properties of the tissuegraft can enhance healing as well.

In other aspects, the tissue grafts can be used in ophthalmologicalapplications (e.g., on-lay grafts ocular surface repair) or urologicalapplications (e.g., facilitate closure of the vas deferens duringvasectomy reversal or facilitate closure of the vas deferens resultingfrom trauma).

In one aspect, the tissue grafts can be used in cranial dura repair andreplacement, in the elimination of a frenum pull, the regeneration oflost patella tissue, the repair of the Schneiderian membrane in thesinus cavity, soft tissue around dental implants, vestibuloplasty, andguided tissue regeneration.

In another aspect, the reinforced tissue grafts can be used in thetreatment of bone defects and bone repair. In one aspect, the reinforcedtissue grafts can be used in dental surgery to provide primary stabilityin mandibular and maxillary horizontal and or vertical guided boneregeneration, repair of dental implants, repair of the sinus, and overmandibular block graft donor sites. In other aspects, the reinforcedtissue grafts can be used in craniofacial surgery, including but notlimited to treatment of bony defects caused from trauma, surgicallycreated bone defects such as burrholes and trephine defects, zygomaticdefects, and orbital defects (FIG. 5). In orthopedic surgery, thereinforced tissue grafts can be used to treat bone defects including butnot limited to open and closed fractures, segmental defects,osteochondral defects, spinal fusion, and other non-load hearingregeneration procedures. In other aspects, the reinforced tissue graftscan be used in the treatment of a segmental long bone defect (FIG. 6).

Depending upon the application of the graft, the graft can be soakedwith a bioactive agent such as a solution composed of naturallyoccurring growth factors sourced from platelet concentrates, eitherusing autologous blood collection and separation products, or plateletconcentrates sourced from expired banked blood; bone marrow aspirate;stem cells derived from concentrated human placental cord blood stemcells, concentrated amniotic fluid stem cells or stem cells grown in abioreactor; or antibiotics. Here, one or more membrane layers of thetissue graft absorb the bioactive agent. Upon application of the wettissue graft with bioactive agent to the wound, the bioactive agent isdelivered to the wound over time.

Although the tissue grafts described herein can be applied directly tothe tissue of a subject, they can also be applied to a wound dressingthat can subsequently be applied to the subject. For example, the wounddressing can be gauze, a bandage or wrap, or any other suitable articlecapable of containing or affixing the tissue graft that can be applieddirectly to a subject.

Preparation of Micronized Composition Example 1

The micronized human amniotic membrane injectable was composed of humanamnion as described above and intermediate layer tissue obtained fromplacenta tissue originated in a hospital, where it is collected during aCesarean section birth. The micronization of the tissue was performedusing a Retsch Oscillating Mill MM400. Phosphate buffer was used as acarrier. The ratio of the injectable was 50 mg/mL. The concentrationratio was 60% (21 mg) amnion and 40% (14 mg) intermediate tissue layerwith 0.70 mL of phosphate buffer. The micronized composition can beadministered as a dermal filler with a 27 gauge needle in the deepdermis region. A suitable dose would be 0.5 cc to 1.0 cc of thecomposition described above.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

A detailed description of suitable cross-linking agents and proceduresis provided in concurrently filed U.S. Patent Application Ser. No.61/683,697 filed as attorney docket number 102741-0250 and entitledPLACENTAL TISSUE GRAFTS MODIFIED WITH A CROSS-LINKING AGENT AND METHODSOF MAKING AND USING THE SAME which application is incorporated herein byreference in its entirety.

A detailed description of micronized placental tissue is provided inconcurrently filed U.S. Patent Application Ser. No. 61/683,698 filed asattorney docket number 102741-0300 and entitled TISSUE GRAFTS COMPOSEDOF MICRONIZED PLACENTAL TISSUE AND METHODS OF MAKING AND USING THE SAMEwhich application is incorporated herein by reference in its entirety.

A detailed description of making and using micronized placental tissueand extracts thereof is provided in concurrently filed U.S. PatentApplication Ser. No. 61/683,700 filed as attorney docket number102741-0400 and entitled MICRONIZED PLACENTAL TISSUE COMPOSITIONS ANDMETHODS OF MAKING AND USING THE SAME which application is incorporatedherein by reference in its entirety.

1. A reinforced tissue graft comprising: a first membrane comprising a placental tissue having a first side and a second side; a biocompatible mesh having a first side and a second side, wherein the first side of the biocompatible mesh is adjacent to the second side of the first membrane; and a second membrane comprising a placental tissue having a first side and a second side, wherein the first side of the second membrane is adjacent to the second side of the biocompatible mesh.
 2. The tissue graft of claim 1, wherein the first membrane comprises amnion, chorion, or a laminate comprising one or more layers of amnion with one or more layers of chorion.
 3. The tissue graft of claim 1, wherein the second membrane comprises amnion, chorion, or a laminate comprising one or more layers of amnion with one or more layers of chorion.
 4. The tissue graft of claim 1, wherein the first membrane comprises modified amnion wherein the modified amnion comprises a first side which is an exposed basement membrane.
 5. The tissue graft of claim 1, wherein the first membrane comprises modified amnion wherein the modified amnion comprises a first side which is an exposed basement membrane and a second side which is an exposed fibroblast layer comprising fibroblast cells.
 6. The tissue graft of claim 1, wherein the second membrane comprises an amnion/chorion laminate, wherein the chorion is adjacent to the second side of the biocompatible mesh.
 7. The tissue graft of claim 6, wherein the amnion comprising an epithelium layer and an intermediate layer, wherein the chorion is adjacent to the intermediate layer.
 8. The tissue graft of claim 6, wherein the amnion comprises a modified amnion comprising an exposed basement membrane and an intermediate layer, wherein the chorion is adjacent to the intermediate layer.
 9. The tissue graft of claim 6, wherein the amnion comprises a modified amnion comprising an exposed basement membrane and an exposed fibroblast layer comprising fibroblast cells, wherein the chorion is adjacent to the exposed fibroblast layer.
 10. The tissue graft of claim 1, wherein the first membrane comprises Wharton's jelly.
 11. The tissue graft of claim 1, wherein the first membrane comprises Wharton's jelly, wherein the epithelium layer is substantially removed.
 12. The tissue graft of claim 1, wherein the first membrane and/or second membrane are crosslinked.
 13. The tissue graft of claim 1, wherein the biocompatible mesh has a pore size from 200 microns to 4,000 microns.
 14. The tissue graft of claim 1, wherein the biocompatible mesh has a plurality of pores spaced from 1,000 microns to 4,500 microns apart as measured from the center of two pores.
 15. The tissue graft of claim 1, wherein the biocompatible mesh has a thickness of 300 microns to 2,000 microns.
 16. The tissue graft of claim 1, wherein the biocompatible mesh comprises a non-resorbable mesh made of a thermoplastic resin, polyethylene, ultra-high weight molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, or any combination thereof.
 17. The tissue graft of claim 1, wherein the biocompatible mesh comprises a resorbable mesh made of polyglycolic acid, poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLA), trimethylenecarbonate (TMC), poly-ε-caprolactone, poly-P-dioxanone, a copolymer of lactide and glycolide (PLGA), polyhydroxy-3-butyrate, collagen, hyaluronic acid, silk, biocellulose, a polysaccharides, poly(DTE carbonate), a polyarylate, blends of PLLA, PLDA, or PLGA with TMC, or any combinations thereof.
 18. The tissue graft of claim 1, wherein the biocompatible mesh is coated on one or both sides with micronized placental tissue.
 19. The tissue graft of claim 18, wherein the biocompatible mesh is structurally homologous.
 20. The tissue graft of claim 19, wherein the biocompatible mesh is wholly comprised of amnion or chorion.
 21. The tissue graft of claim 18, wherein the biocompatible mesh is structurally heterologous.
 22. The tissue graft of claim 21, wherein the biocompatible mesh is comprised of amnion, chorion or combinations thereof.
 23. The tissue graft of claim 18, wherein the micronized placental tissue is applied to the tissue graft by wetting the biocompatible mesh to render it absorbent and contacting the biocompatible mesh with a surface having micronized placental tissue deposited thereon.
 24. A wound dressing comprising the tissue graft of claim
 1. 25-93. (canceled) 